Heat shock protein-based vaccines and immunotherapies

ABSTRACT

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein the subject is administered an effective amount of at least one heat shock protein in combination with one or more defined target antigens. These methods and compositions may be used in the treatment of infectious eases and cancers.

The invention described herein was made in the course of work under NIHCore Grant No. CA 08748. The United States government may have certainrights in this invention.

INTRODUCTION

The present invention relates to methods and compositions for inducingan immune response in a subject, wherein the subject is administered aneffective amount of at least one heat shock protein in combination withone or more defined target antigens. These methods and compositions maybe used in the treatment of infectious diseases and cancers.

BACKGROUND OF THE INVENTION

Heat shock proteins were originally observed to be expressed inincreased amounts in mammalian cells which were exposed to suddenelevations of temperature, while the expression of most cellularproteins is significantly reduced. It has since been determined thatsuch proteins are produced in response to various types of stress,including glucose deprivation. As used herein, the term “heat shockprotein” will be used to encompass both proteins that are expresslylabeled as such as well as other stress proteins, including homologs ofsuch proteins that are expressed constitutively (i.e., in the absence ofstressful conditions). Examples of heat shock proteins include BiP (alsoreferred to as grp78), hsp/hsc70, gp96 (grp94), hsp60, hsp40 and hsp90.

Heat shock proteins have the ability to bind other proteins in theirnon-native states, and in particular to bind nascent peptides emergingfrom ribosomes or extruded into the endoplasmic reticulum. Hendrick andHartl., Ann. Rev. Biochem. 62:349-384 (1993); Hartl., Nature 381:571-580(1996). Further, heat shock proteins have been shown to play animportant role in the proper folding and assembly of proteins in thecytosol, endoplasmic reticulum and mitochondria; in view of thisfunction, they are referred to as “molecular chaperones”. Frydman etal., Nature 370: 111-117 (1994); Hendrick and Hartl., Ann. Rev. Biochem.62:349-384 (1993); Hartl, Nature 381:571-580 (1996).

For example, the protein BiP, a member of a class of heat shock proteinsreferred to as the hsp70 family, has been found to bind to newlysynthesized, unfolded p immunoglobulin heavy chain prior to its assemblywith light chain in the endoplasmic reticulum. Hendershot et al., J.Cell Biol. 104:761-767 (1987). Another heat shock protein, gp96, is amember of the hsp90 family of stress proteins which localize in theendoplasmic reticulum. Li and Srivastava, EMBO J. 12:3143-3151 (1993);Mazzarella and Green, J. Biol. Chem. 262:8875-8883 (1987). It has beenproposed that gp96 may assist in the assembly of multi-subunit proteinsin the endoplasmic reticulum. Wiech et al., Nature 358:169-170 (1992).

It has been observed that heat shock proteins prepared from tumors inexperimental animals were able to induce immune responses in atumor-specific manner; that is to say, heat shock protein purified froma particular tumor could induce an immune response in an experimentalanimal which would inhibit the growth of the same tumor, but not othertumors. Srivastava and Maki, 1991, Curr. Topics Microbial. 167:109-123(1991). The source of the tumor-specific immunogenicity has not beenconfirmed. Genes encoding heat shock proteins have not been found toexhibit tumor-specific DNA polymorphism. Srivastava and Udono, Curr.Opin. Immunol. 6:728-732 (1994). High resolution gel electrophoresis hasindicated that gp96 may be heterogeneous at the molecular level. Feldwegand Srivastava, Int. J. Cancer 63: 310-314 (1995). Evidence suggeststhat the source of heterogeneity may be populations of small peptidesadherent to the heat shock protein, which may number in the hundreds.Id. It has been proposed that a wide diversity of peptides adherent totumor-synthesized heat shock proteins may render such proteins capableof eliciting an immune response in subjects having diverse HLAphenotypes, in contrast to more traditional immunogens which may besomewhat HLA-restricted in their efficacy. Id.

Recently, Nieland et al. (Proc. Natl. Acad. Sci. U.S.A. 93:6135-6139(1996)) identified an antigenic peptide containing a cytotoxic Tlymphocyte (CTL) vesicular stomatitis virus (VSV) epitope bound to gp96produced by VSV-infected cells. Neiland's methods precluded theidentification of any additional peptides or other compounds which mayalso have bound to gp96, and were therefore unable to furthercharacterize higher molecular weight material which was bound to gp96and detected by high pressure liquid chromatography.

It has been reported that a synthetic peptide comprising multipleiterations of NANP (Asp Ala Asp Pro) malarial antigen, chemicallycross-linked to glutaraldehyde-fixed mycobacterial hsp65 or hsp70, wascapable of inducing antibody formation (i.e., a humoral response) inmice in the absence of any added adjuvant; a similar effect was observedusing heat shock protein from the bacterium Escherichia coli. DelGuidice, Experientia 50:1061-1066 (1994); Barrios et al., Clin. Exp.Immunol. 98:224-228 (1994); Barrios et al., Eur. J. Immunol.22:1365-1372 (1992). Cross-linking of synthetic peptide to heat shockprotein and possibly glutaraldehyde fixation was required for antibodyinduction. Barrios et al., Clin. Exp. Immunol. 98:229-233.

It has now been discovered, according to the present invention, thatheat shock protein may be combined with target antigen and used toinduce an immune response which includes a cytotoxic cellular component,i.e., a cellular response.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for inducingan immune response in a subject, wherein at least one heat shock proteinin combination with one or more defined target antigens is administeredto the subject.

Unlike prior disclosures relating to heat shock protein associated withan undefined population of potential antigens which have beenrestricted, in their immunogenic effect, to a single tumor, the presentinvention provides for methods and compositions which combine heat shockprotein with a defined target antigen which may be selected on the basisthat it is immunogenic in diverse occurrences of a neoplastic orinfectious disease, or because it has been identified, in an individualinstance, as being particularly immunogenic. Further, because the use ofone or more defined target antigen permits more control over the immuneresponse elicited, it may avoid the induction of an undesirable immuneresponse.

In alternative embodiments of the invention, the target antigen may beeither (i) an antigen which itself binds to the heat shock protein; or(ii) a hybrid antigen comprising an immunogenic domain as well as a heatshock protein-binding domain. The immunogenic domain may be an entireprotein or peptide antigen, or may be only a portion of the selectedantigen, for example a selected epitope of the antigen. In specific,nonlimiting embodiments of the invention, the heat shock protein bindingdomain may comprise a peptide having the sequence:

His Trp Asp Phe Ala Trp Pro Trp [SEQ. ID NO. 1]

The present invention provides for methods of administering such heatshock protein/target antigen compositions comprising (i) combining oneor more heat shock protein with one or more target antigens in vitro,under conditions wherein binding of target antigen to heat shock proteinoccurs to form a target antigen/heat shock protein complex; and (ii)administering the target antigen, bound to heat shock protein, in aneffective amount to a subject in need of such treatment.

Alternatively, heat shock protein/target antigen combinations of theinvention may be administered to a subject by introducing nucleic acidencoding the heat shock protein and the target antigen into the subjectsuch that the heat shock protein and target antigen bind in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the induction of a cellular immune response using hybridpeptide antigens in accordance with the invention;

FIG. 2 shows the induction of a cellular immune response using hybridpeptide antigens in accordance with the invention;

FIG. 3 shows the induction of a cellular immune response using hybridpeptide antigens in accordance with the invention;

FIG. 4 shows the induction of a cellular immune response using hybridpeptide antigens in accordance with the invention;

FIGS. 5A and 5B shows the results of control experiments in which hybridpeptide or Ova-peptide and heat shock protein were administeredindividually to EL4 cells;

FIG. 6 shows co-elution of hybrid peptides and heat shock proteins froma column, demonstrating binding of the polypeptides to the heat shockprotein;

FIG. 7 shows the co-elution of ¹²⁵I-OVA-BiP with BiP in the presence andabsence of ATP;

FIG. 8 shows the killing efficacy of T-cells primed with variouscombinations of antigens and heat shock proteins on EL4 cells pulsedwith antigen; and

FIG. 9 shows the killing efficacy of T-cells primed with variousconcentrations of antigens and heat shock proteins on EG7 lymphomacells.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of description, and not by way of limitation,the detailed description is divided into the following subsections:

(i) heat shock proteins;

(ii) target antigens; and

(iii) methods of administration.

Heat Shock Proteins

The term “heat shock protein”, as used herein, refers to any proteinwhich exhibits increased expression in a cell when the cell is subjectedto a stress. In preferred nonlimiting embodiments, the heat shockprotein is originally derived from a eukaryotic cell; in more preferredembodiments, the heat shock protein is originally derived from amammalian cell. For example, but not by way of limitation, heat shockproteins which may be used according to the invention include BiP (alsoreferred to as grp78), hsp/hsc70, gp96(grp94), hsp60, hsp40, and hsp90.Especially preferred heat shock proteins are BiP, gp96, and hsp70, asexemplified below. Naturally occurring or recombinantly derived mutantsof heat shock proteins may also be used according to the invention. Forexample, but not by way of limitation, the present invention providesfor the use of heat shock proteins mutated so as to facilitate theirsecretion from the cell (for example having mutation or deletion of anelement which facilitates endoplasmic reticulum recapture, such as KDELor its homologs; such mutants are described in concurrently filed PCTApplication No. ______ (Attorney Docket No. MSK.P-018), which isincorporated herein by reference.

For embodiments of the invention wherein heat shock protein and targetantigen are directly administered to the subject in the form of aprotein/peptide complex, the heat shock protein may be prepared, usingstandard techniques, from natural sources, for example as described inFlynn et al., Science 245: 385-390 (1989), or using recombinanttechniques such as expression of a heat shock encoding vector in asuitable host cell such as a bacterial, yeast or mammalian cell. Ifpre-loading of the heat shock protein with peptides from the hostorganism is a concern, the heat shock protein can be incubated with ATPand then repurified. Nonlimiting examples of methods for preparingrecombinant heat shock proteins are set forth below.

A nucleic acid encoding a heat shock protein may be operatively linkedto elements necessary or desirable for expression and then used toexpress the desired heat shock protein as either a means to produce heatshock protein for use in a protein vaccine or, alternatively, in anucleic acid vaccine. Elements necessary or desirable for expressioninclude, but are not limited to, promoter/enhancer elements,transcriptional start and stop sequences, polyadenylation signals,translational start and stop sequences, ribosome binding sites, signalsequences and the like. For example, but not by way of limitation, genesfor various heat shock proteins have been cloned and sequenced,including, but not limited to, gp96, human: Genebank Accession No.X15187; Maki et al., Proc. Nat'l Acad. Sci. 87: 5658-5562 (1990), mouse:Genebank Accession No. M16370; Srivastava et al., Proc. Nat'l Acad. Sci.84:3807-3811 (1987)); BiP, mouse: Genebank Accession No. U16277; Haas etal., Proc. Nat'l Acad. Sci. U.S.A. 85: 2250-2254 (1988), human: GenebankAccession No. M19645; Ting et al., DNA 7: 275-286 (1988); hsp70, mouse:Genebank Accession No. M35021; Hunt et al., Gene 87: 199-204 (1990),human: Genebank Accession No. M24743; Hunt et al., Proc. Nat'l Acad.Sci. U.S.A. 82: 6455-6489 (1995); and hsp40 human: Genebank AccessionNo. D49547; Ohtsuka K., Biochem Biophys. Res. Commun. 197: 235-240(1993).

Target Antigens

A target antigen, according to the invention, may be either (i) anantigen which itself binds to the heat shock protein; or (ii) a hybridantigen comprising an immunogenic domain as well as a heat shockprotein-binding domain. Thus, the target antigen serves at least twofunctions, namely (I) it contains an epitope capable of inducing thedesired immune response; and (ii) it is capable of physically binding toits partner heat shock protein. Of note, the term “physically binds”indicates that the target antigen and heat shock protein exhibit aphysical interaction which permits the adherence of one to the other forat least a transient period of time; of note, the binding need not, andin most embodiments of the invention should not, be irreversible.

In certain embodiments, an antigen capable of inducing the desiredimmune response may be found to be inherently capable of binding to apartner heat shock protein. In other embodiments, it may be necessary ordesirable to link an immunogenic antigen to one or more other compoundsso as to create a hybrid antigen which contains both an immunogenicdomain as well as a heat shock protein binding domain. In suchcircumstances, a compound which is, itself, an immunogenic antigen maybe linked to a compound which is, itself, capable of binding to a heatshock protein. Alternatively, the linkage of two or more compounds whichindividually lack either functionality may give rise to the desiredimmunogenic and binding characteristics.

The term “antigen” as used herein, refers to a compound which may becomposed of amino acids, carbohydrates, nucleic acids or lipidsindividually or in any combination.

The term “target antigen”, as used herein, refers to a compound whichbinds to one or more heat shock proteins and which is representative ofthe immunogen toward which an immune response is desirably directed. Forexample, where the immunogen is an influenza virus, the target antigenmay be a peptide fragment of the matrix protein of the influenza virus.As used herein, the term “immunogen” is applied to the neoplastic cell,infected cell, pathogen, or component thereof, towards which an immuneresponse is to be elicited, whereas the target antigen is a portion ofthat immunogen which can provoke the desired response and whichinherently or through engineering binds to one or more heat shockproteins. In particular, the target antigen is selected to elicit animmune response to a particular disease or pathogen, including peptidesobtained from MHC molecules, mutated DNA gene products, and direct DNAproducts such as those obtained from tumor cells.

While the invention may be applied to any type of immunogen, immunogensof particular interest are those associated with, derived from, orpredicted to be associated with a neoplastic disease, including but notlimited to a sarcoma, a lymphoma, a leukemia, or a carcinoma, and inparticular, with melanoma, carcinoma of the breast, carcinoma of theprostate, ovarian carcinoma, carcinoma of the cervix, colon carcinoma,carcinoma of the lung, glioblastoma, astrocytoma, etc. Further,mutations of tumor suppressor gene products such as p53, or oncogeneproducts such as ras may also provide target antigens to be usedaccording to the invention.

In further embodiments, the immunogen may be associated with aninfectious disease, and, as such, may be a bacterium, virus, protozoan,mycoplasma, fungus, yeast, parasite, or prion. For example, but not byway of limitation, the immunogen may be a human papilloma virus (seebelow), a herpes virus such as herpes simplex or herpes zoster, aretrovirus such as human immunodeficiency virus 1 or 2, a hepatitisvirus, an influenza virus, a rhinovirus, respiratory syncytial virus,cytomegalovirus, adenovirus, Mycoplasma pneumoniae, a bacterium of thegenus Salmonella, Staphylococcus, Streptococcus, Enterococcus,Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, amalarial parasite, Trypanosoma cruzi, etc.

Immunogens may be obtained by isolation directly from a neoplasm, aninfected cell, a specimen from an infected subject, a cell culture, oran organism culture, or may be synthesized by chemical or recombinanttechniques. Suitable antigenic peptides, particularly for use in ahybrid antigen, for use against viruses, bacteria and the like can bedesigned by searching through their sequences for MHC class I restrictedpeptide epitopes containing HLA binding sequences such as but notlimited to HLA-A2 peptide binding sequences:

[SEQ ID No. 2] Xaa (Leu/Met) XaaXaaXaa (Val/Ile/Leu/Thr) XaaXaa(Val/Leu),

Rammensee et al., Immunogenetics 41: 178-223 (1995),

Xaa (Leu/Met) XaaXaaXaaXaaXaaXaaVal, [SEQ ID No. 3]

Tarpey, et al Immunology 81: 222-227 (1994),

Xaa(Val/Gln)XaaXaaXaaXaaXaaXaaLeu [SEQ ID No. 28]Barouch et al., J. Exp. Med. 182: 1847-1856 (1995).

It may also be desirable to consider the type of immune response whichis desired. For example, under certain circumstances, a humoral immuneresponse may be appropriate. In other cases, and indeed where an immuneresponse directed toward neoplastic cells or infected cells is sought tobe elicited, a cellular immune response is particularly desirable.Accordingly, particular epitopes associated with the activation of Bcells, T helper cells, or cytotoxic T cells may be identified andselected for incorporation into the target antigen.

It may also be desirable to utilize target antigen associated with anautoimmune disease or allergy. Such a target antigen may beadministered, together with one or more heat shock proteins, in anamount sufficient to be tolerogenic or to inhibit a pre-existing immuneresponse to the target antigen in a subject. The amount of heat shockprotein required to inhibit the immune response is expected to besubstantially greater than the amount required for stimulation.

Although the size of target antigen may vary depending upon the heatshock protein used, in nonlimiting embodiments of the invention, thetarget antigen may be the size of a peptide having between 4 and 500 ofamino acid residues, and preferably be the size of a peptide havingbetween 4 and 100, most preferably 7 and 20 amino acid residues. Assuch, it may be desirable to produce a fragment of an immunogen to serveas a target antigen, or, alternatively, to synthesize a target antigenby chemical or recombinant DNA methods. In some instances, however, animmunogen may, in intact form, serve as a target antigen.

Based on the foregoing considerations, a target antigen may be prepared,and then tested for its ability to bind to heat shock protein. In someinstances, binding of target antigen to a particular heat shock proteinmay be facilitated by the presence of at least one other protein, whichmay be a heat shock protein.

For example, binding of target antigen to a heat shock protein may beevaluated by labeling the target antigen with a detectable label, suchas a radioactive, fluorescent, enzymatic or pigmented label, combiningthe target antigen with heat shock protein under conditions which wouldbe expected to permit binding to occur, and then isolating the heatshock protein while removing any unbound target antigen, and determiningwhether any labeled target antigen had adhered to the heat shockprotein. As a specific example, and not by way of limitation, theability of a target antigen to bind to BiP heat shock protein may beevaluated by combining 2 μg BiP with up to about 1150 pmole ofradioactively labeled target antigen in buffer containing 50 mM Tris HCl(pH 7.5), 200 mM NaCl, and 1 mM Na₂EDTA, in a final volume of 50 μl, for30 minutes at 37 degrees Centigrade. Unbound target antigen may then beremoved from bound BiP-target antigen by centrifugation at long bydesalting through a 1 ml Sephadex-G column for 2 minutes. Penefsky, J.Biol. Chem. 252:2891 (1977). To prevent binding to the resin, columnsmay first be treated with 100 μl of bovine serum albumin in the samebuffer and centrifuged as above. Bound target antigen may then bequantitated by liquid scintillation counting. See Flynn et al., Science245:385-390 (1989).

Because ATP hydrolysis drives the release of peptides from many knownheat shock proteins, the amount of ATPase activity may often be used toquantitate the amount of target antigen binding to heat shock protein.An example of how such an assay may be performed is set forth in Flynnet al., Science 245:385-390 (1990).

If a particular immunogen or a fragment thereof does not satisfactorilybind to a heat shock protein, then that immunogen or fragment may belinked to another compound so as to create a heat shock protein-bindingdomain thereby constructing a hybrid antigen. The heat shockprotein-binding domain is selected so that the hybrid peptide will bindin vitro to a heat shock protein such as BiP, hsp70, gp96, or hsp90,alone or in combination with accessory heat shock proteins such ashsp40, or hsp60. Peptides which fulfill this criterion may be identifiedby panning libraries of antigens known to bind well to one or more heatshock proteins as described in Blond-Elguindi et al., Cell 75:717-728(1993). Using this technique, Blond-Elguindi have concluded that theheat shock protein BiP recognizes polypeptides that contain a heptamericregion having the sequence

Hy (Trp/X) HyXHyXHywhere Hy represents a hydrophobic amino acid residue, particularlytryptophan, leucine or phenylalanine, and X is any amino acid. Highaffinity heat-shock protein-binding sequences incorporating this motifinclude:

His Trp Asp Phe Ala Trp Pro Trp; [Seq. ID No. 1] and Phe Trp Gly Leu TrpPro Trp Glu. [Seq. ID No. 4]

Other heat shock protein binding motifs have also been identified. Forexample, Auger et al. Nature Medicine 2:306-310 (1996) have identifiedtwo pentapeptide binding motifs

Gln Lys Arg Ala Ala [SEQ ID No. 5] and Arg Arg Arg Ala Ala [Seq. ID No.6]in HLA-DR types associated with rheumatoid arthritis which bind to heatshock proteins. Heat shock binding motifs have also been identified asconsisting of seven to fifteen residue long peptides which are enrichedin hydrophobic amino acids. Flynn et al., Science 245: 385-390 (1989);Gragerov et al., J. Molec. Biol. 235: 848-854 (1994).

The hybrid antigen of the invention incorporates one immunogenic domainand one heat shock protein-binding domain, optionally separated by ashort peptide linker. The hybrid peptide of the invention may besynthesized using chemical peptide synthesis methods or it can besynthesized by expression of a nucleic acid construct containing linkedsequences encoding the antigenic and heat shock protein-binding domains.One suitable technique utilizes initial separate PCR amplificationreactions to produce separate DNA segments encoding the two domains,each with a linker segment attached to one end, followed by fusion ofthe two amplified products in a further PCR step. This technique isreferred to as linker tailing. Suitable restriction sites may also beengineered into regions of interest, after which restriction digestionand ligation is used to produce the desired hybrid peptide-encodingsequence.

Methods of Administration

The heat shock protein/target antigen combinations of the invention maybe administered to a subject using either a protein-based or nucleicacid vaccine, so as to produce, in the subject, an amount of heat shockprotein/target antigen complex which is effective in inducing atherapeutic immune response in the subject.

The subject may be a human or nonhuman subject.

The term “therapeutic immune response”, as used herein, refers to anincrease in humoral and/or cellular immunity, as measured by standardtechniques, which is directed toward the target antigen. Preferably, butnot by way of limitation, the induced level of humoral immunity directedtoward target antigen is at least four-fold, and preferably at least16-fold greater than the levels of the humoral immunity directed towardtarget antigen prior to the administration of the compositions of thisinvention to the subject. The immune response may also be measuredqualitatively, wherein by means of a suitable in vitro assay or in vivoan arrest in progression or a remission of neoplastic or infectiousdisease in the subject is considered to indicate the induction of atherapeutic immune response.

Specific amounts of heat shock protein/target antigen administered maydepend on numerous factors including the immunogenicity of theparticular vaccine composition, the immunocompetence of the subject, thesize of the subject and the route of administration. Determining asuitable amount of any given composition for administration is a matterof routine screening.

In specific nonlimiting embodiments of the invention, it may bedesirable to include more than one species of heat shock protein, and/ormore than one target antigen, in order to optimize the immune response.Such an approach may be particularly advantageous in the treatment ofcancer or in the treatment of infections characterized by the rapiddevelopment of mutations that result in evasion of the immune response.

In other specific nonlimiting embodiments of the invention, in order topromote binding among members of each heat shock protein/target antigenpair, the ratio of heat shock protein to target antigen may preferablybe 1:2 to 1:200. Higher relative levels of antigen are suitable toenhance binding to the heat shock protein.

According to still further specific but nonlimiting embodiments of theinvention, the target antigen is not chemically cross-linked to the heatshock protein.

Compositions comprising target antigen/heat shock protein as set forthabove are referred to herein as “vaccines”. The term vaccine is used toindicate that the compositions of the invention may be used to induce atherapeutic immune response.

A vaccine composition comprising one or more heat shock proteins and oneor more target antigens in accordance with the invention may beadministered cutaneously, subcutaneously, intravenously,intramuscularly, parenterally, intrapulmonarily, intravaginally,intrarectally, nasally or topically. The vaccine composition may bedelivered by injection, particle bombardment, orally or by aerosol.

Incubation of heat shock proteins in solution with the target antigen issufficient to achieve loading of the antigen onto the heat shock proteinin most cases. It may be desirable in some cases, however, to add agentswhich can assist in the loading of the antigen.

Incubation with heating of the heat shock protein with the targetantigen will in general lead to loading of the antigen onto the heatshock protein. In some cases, however, it may be desirable to addadditional agents to assist in the loading. For example, hsp40 canfacilitate loading of peptides onto hsp70. Minami et al., Gen. Biol.Chem. 271: 19617-19624 (1996). Denaturants such as guanidinium HCl orurea can be employed to partially and reversibly destabilize the heatshock protein to make the peptide binding pocket more accessible to theantigen.

Vaccine compositions in accordance with the invention may furtherinclude various additional materials, such as a pharmaceuticallyacceptable carrier. Suitable carriers include any of the standardpharmaceutically accepted carriers, such as phosphate buffered salinesolution, water, emulsions such as an oil/water emulsion or atriglyceride emulsion, various types of wetting agents, tablets, coatedtablets and capsules. An example of an acceptable triglyceride emulsionuseful in intravenous and intraperitoneal administration of thecompounds is the triglyceride emulsion commercially known asIntralipid®. Typically such carriers contain excipients such as starch,milk, sugar, certain types of clay, gelatin, stearic acid, talc,vegetable fats or oils, gums, glycols, or other known excipients. Suchcarriers may also include flavor and color additives or otheringredients.

The vaccine composition of the invention may also include suitablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions may be in the form of liquid or lyophilizedor otherwise dried formulations and may include diluents of variousbuffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), solubilizing agents (e.g. glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexing withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc. or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. The choice of compositions will depend on the physical andchemical properties of the vaccine. For example, a product derived froma membrane-bound form of a protein may require a formulation containingdetergent. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g. fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g. poloxamers or poloxamines) and coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhances forvarious routes of administration, including intramuscular, parenteral,pulmonary, nasal and oral.

As an alternative to direct administration of the heat shock protein andtarget antigen, one or more polynucleotide constructs may beadministered which encode heat shock protein and target antigen inexpressible form. The expressible polynucleotide constructs areintroduced into cells in the subject using ex vivo or in vivo methods.Suitable methods include injection directly into tissue and tumors,transfecting using liposomes (Fraley et al., Nature 370:111-117 (1980)),receptor-mediated endocytosis (Zatloukal, et al., Ann. NY Acad. Sci.660:136-153 (1992)); particle bombardment-mediated gene transfer(Eisenbraun et al., DNA & Cell Biol. 12:792-797 (1993)) and transfectionusing peptide presenting bacteriophage. Barry et al. Nature Medicine 2:299-305 (1996). The polynucleotide vaccine may also be introduced intosuitable cells in vitro which are then introduced into the subject.

To construct an expressible polynucleotide, a region encoding the heatshock protein and/or target antigen is prepared as discussed above andinserted into a mammalian expression vector operatively linked to asuitable promoter such as the SV40 promoter, the cytomegalovirus (CMV)promoter or the Rous sarcoma virus (RSV) promoter. The resultingconstruct may then be used as a vaccine for genetic immunization. Thenucleic acid polymer(s) could also be cloned into a viral vector.Suitable vectors include but are not limited to retroviral vectors,adenovirus vectors, vaccinia virus vectors, pox virus vectors andadenovirus-associated vectors. Specific vectors which are suitable foruse in the present invention are pCDNA3 (In-Vitrogen), plasmid AHS(which contains the SV40 origin and the adenovirus major late promoter).pRC/CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927(1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSRα(DNAX, Palo Alto, Calif.).

EXAMPLE 1 Preparation of Hybrid Peptides

Hybrid peptides containing a BiP-binding domain(His-Trp-Asp-Phe-Ala-Trp-Pro-Trp; SEQ ID NO: 1) and an OVA antigenicdomain (Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu; SEQ ID NO:7) separated by atripeptide linker (gly-ser-gly) were synthesized. Peptides were producedin both orientations, OVA-BiP-binding domain and BiP-binding domain OVAas follows:

[SEQ ID NO: 8] Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Gly-Ser-Gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp and [SEQ ID NO. 9]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-Gly-Ser-Gly-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu.

EXAMPLE 2

Purified mouse cytosolic hsp70 was prepared from E. coli DH5α cellstransformed with pMS236 encoding mouse cytosolic hsp70. The cells weregrown to an optical density (600 nm) of 0.6 at 37° C., and expressionwas induced by the addition of IPTG to a final concentration of 1 mM.Cells were harvested by centrifugation 2 to 5 hours post induction andthe pellets were resuspended to 20 mL with Buffer A (20 mM Hepes pH 7.0,25 mM KCl, 1 mM DTT, 10 mM (NH₄)₂SO₄, 1 mM PMSF). The cells were lysedby passing three times through a French press. The lysate was cleared bya low speed spin, followed by centrifugation at 100,000×G for 30minutes. The cleared lysate was applied to a Pharmacia XK26 columnpacked with 100 mL DEAE Sephacel and equilibrated with Buffer A at aflow rate of 0.6 cm/min. The column was washed to stable baseline withBuffer A and eluted with Buffer A adjusted to 175 mM KCl. The eluate wasapplied to a 25 mL ATP-agarose column (Sigma A2767), washed to baselinewith Buffer A, and eluted with Buffer A containing 1 mM MgATPpreadjusted to pH 7.0. EDTA was added to the eluate to a finalconcentration of 2 mM. The eluate which contained essentially pure hsp70was precipitated by addition of (NH₄)₂SO₄ to 80% saturation. Theprecipitate was resuspended in Buffer A containing 1 mM MgCl₂ anddialyzed against the same buffer with multiple changes. The purifiedhsp70 was frozen in small aliquots at −70° C.

EXAMPLE 3

The purified hsp70 was combined with the synthesized peptides and usedfor immunization. To form the hsp70/peptide mixtures, approximately 15ug (21.5 uM) hsp70 was combined with 5 ug of Ova-peptide (0.5 mM, SEQ.ID. NO: 5) or 10 ug (0.5 mM) hybrid peptide (SEQ. ID NOS: 6 and 7) weremixed on ice to a final volume of 10 μl in Buffer B (finalconcentration: 20 mM Hepes pH 7.0, 150 mM KCl, 10 mM (NH₄)₂SO₄, 2 mMMgCl₂ and 2 mM MgATP, pH 7.0). The mixtures were incubated for 30minutes at 37° C. and then used for in vivo immunizations.

C57BL/6 mice were immunized intradermally once a week for a total of twoweeks with 10 μL of one of the mixtures described above or with amixture of TiterMax® (Vaxell, Norcross, Ga.) and Ova-peptide (5 μg). Oneweek after the second immunization, spleen cells were removed andmononuclear cells (6-8×10⁷) were cultured with 3×10⁶ γ-irradiated (3000rad) stimulator cells. The stimulator cells were obtained from naivemice that had been sensitized in vitro with Ova-peptide (10 mg/ml) for30 minutes at room temperature, washed and irradiated at 3000 rads.

Cytotoxicity of spleen cells from vaccinated mice were assayed onOva-peptide pulsed EL4 cells in an 18-hour chromium release assay. CTLwere generated by culturing in vivo immunized spleen cells for 5 days ata concentration of 10⁶ cells/mL in RPMI medium, 10% FCS, penicillin,streptomycin and 2 mM L-glutamine, together with 3×10⁶ γ-irradiated(3,000 rad) stimulator cells/mL. Target cells were prepared by culturingcells for 1 hour in the presence of 250 μCi of ⁵¹Cr sodium chromate(DuPont, Boston, Mass.) in Tris-phosphate buffer, pH 7.4 at 37° C. for60 minutes. After washing, 10⁴ ⁵¹Cr-labeled target cells were mixed witheffector lymphocytes to yield several different effector/target (E/T)ratios and were incubated for 18 hours. Supernatants were harvested andthe radioactivity was measured in a gamma counter. Percent specificlysis was calculated as: 100×[(cpm release by CTL−cpm spontaneousrelease)/(cpm maximal release−cpm spontaneous release)]. Maximalresponse was determined by addition of 1% Triton X-100. Spontaneousrelease of all target in the absence of effector cells was less than 25%of the maximal release.

As shown in FIG. 1, the combination of Hsp70 and the hybrid peptide ofeither orientation (hsp7+BiP-OVA or hsp70+OVA-BiP) evoked a higherimmune response as measured by specific lysis of cells than the hsp70 orTiterMax® adjuvant plus Ova-peptide alone.

EXAMPLE 4

The assay of Example 3 was repeated using CTL cell lines which had beenmaintained by stimulation with irradiated stimulators, syngeneic splenicfeeder cells plus T cell growth factors for a period of two weeks. Asshown in FIG. 2, the combination of hsp70 and the hybrid peptide ofeither orientation (hsp70+BiP-OVA or hsp7+OVA-BiP) evoked a higherimmune response as measured by specific lysis of cells than the hsp70 orTiterMax® adjuvant plus Ova-peptide alone. Thus, the immune responseelicited by the hybrid peptides persisted through additional passagesand can be maintained over a period of time.

EXAMPLE 5

The experiment of Example 2 was repeated for the combinations of hsp70plus BiP-OVA and TiterMax® plus Ova-peptide using only a singleimmunization one week before removal of the spleen cells. As shown inFIG. 3, the single immunization with either composition was effective ineliciting a cellular immune response.

EXAMPLE 6

The assay of Example 3 was repeated using mixtures of TiterMax® withOva-peptide or the hybrid peptides of Example 1. As shown in FIG. 4, nosignificant difference was observed between the Ova-peptide and hybridpeptides demonstrating the specificity of the effect when hybridpeptides are used in association with the heat shock protein.

EXAMPLE 7

FIGS. 5A and 5B show the results when the procedure of Example 3 wasrepeated immunizing the mice with hsp70 alone, OVA-peptide alone,Ova-BiP alone or Bip-Ova alone. As shown, the results in all cases werethe same when the cells were pulsed with Ova-peptide (FIG. 5A) and whenthey had not been pulsed. (FIG. 5B). This demonstrates that the responseis the result of the combination of the mixture of the antigen(Ova-peptide or hybrid peptide) and the heat shock protein and not toany of the components individually.

EXAMPLE 8

¹⁴C-labeled OVA-BiP was prepared by alkylation of OVA-BiP with¹⁴C-formaldehyde. 0.9 mg of OVA-BiP in 300 uL 10% DMSO/water was addedto 175 μl of ¹⁴C-formaldehyde (62 μCi) and immediately 50 uL of freshlymade up 200 mM NaCNBH₃ was added. The reaction was mixed and left at 25°C. for 3 hours. The labeled peptide was repurified by reverse phase HPLCon a C-4 column in a 15 minute 0-100% acetonitrile (0.1% TFA) gradient.

The ability of the OVA-BiP peptide to bind to heat shock proteins wasmeasured by incubating 100 μM (5 μg) ¹⁴C-labeled OVA-BiP with 50 μg ofBiP (prepared as in example 11), hsp70 (as prepared in Example 2) orgrp96 (prepared as in Example 10) in a final volume of 20 μl of buffer(50 mM Mops, pH 7.2., 200 m mM NaCl, 5 mM MgAcetate) at 37° C. for 30minutes. The samples were then spun down (5 minutes in a microfuge) andloaded onto a 17 cm long Sephacryl S-300 column equilibrated in bindingbuffer (50 mM Mops, pH 7.2., 200 mM NaCl, 5 mM MgAcetate) and fractionswere collected dropwise. 50 μl of each ˜225 μl fraction was counted inscintillation liquid. 10 μl of each fraction was also run on a 12%SDS-PAGE reducing gel. FIG. 6 shows the radioactivity detected in eachfraction eluted from the column, together with the center of the peak ofheat shock protein as determined by SDS-PAGE. As shown, a significantamount of radioactivity elutes with BiP and hsp70, thus providingevidence that the hybrid peptide binds to these two heat shock proteins.The result for gp96 is unclear because the peak at fraction 11 (whichmay represent an aggregation phenomenon) and the gp96 peak (fraction 14)elute close together on the column used.

EXAMPLE 9

To prepare ¹²⁵I-OVA-BiP, 250 μCi of monoiodinated Bolton-Hunter reagentwas transferred into a stoppered vial and the solvent in which it wasdissolved was evaporated with a gentle stream of argon gas. To the driedreagent 222 μL of 4.5 mg/mL OVA-BiP in 100 mM NaBO₃, pH 8, 9, 10% DMSOwas added. The reaction was mixed and incubated at 25° C. for 45 minutesand continued at 4° C. for a further hour. The labeled peptide wasrepurified by reverse phase HPLC on a C-4 column in a 20 minute, 0-100%acetonitrile (0.1% TFA) gradient.

The iodinated OVA-BiP was combined with BiP in substantially the samemanner as the heat shock proteins in Example 7, except that since theiodinated peptide was at a very low concentration, 1 μl (approx 32 ng)of labeled peptide was mixed with 5 μg of unlabeled peptide and this wasincubated with 50 μg of BiP in 20 μL of binding buffer. To observeATP-mediated peptide release, ATP was added to a final concentration of2 mM after the 30 minute incubation and incubated for a further 5minutes prior to spinning. These samples were run on the same column asabove, but equilibrated in binding buffer supplemented with 2 mM ATP.

FIG. 7 shows the elution profile for a mixture of the ¹²⁵I-OVA-BiP andBiP in the presence and absence of 2 mM ATP. As shown, addition of ATPcauses the release of the hybrid peptide from the BiP. This isconsistent with the observation that ATP mediates release of boundproteins or polypeptides from heat shock proteins.

EXAMPLE 10

Hybrid peptides for use in a vaccine in accordance with the inventionagainst human papilloma virus are prepared using a peptide synthesizeras follows:

E7 (Type 11) -BiP [SEQ ID No. 10]Leu-Leu-Leu-Gly-Thr-Leu-Asn-Ile-Val-gly-ser-gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Type 11) [SEQ ID No. 11]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Leu-Leu-Leu-Gly-Thr-Leu-Asn-Ile-Val E7 (Type 16) -BiP [SEQ ID No. 12]Leu-Leu-Met-Gly-Thr-Leu-Gly-Ile-Val-gly-ser-gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Type 16) [SEQ ID No. 13]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Leu-Leu-Met-Gly-Thr-Leu-Gly-Ile-Val E7 (Type 18) -BiP [SEQ ID No. 14]Thr-Leu-Gln-Asp-Ile-Val-Leu-His-Leu-gly-ser-gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Tvpe 18) [SEQ ID No. 15]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Thr-Leu-Gln-Asp-Ile-Val-Leu-His-Leu E7.1 (Type 6b)-BiP [SEQ ID No. 16]Gly-Leu-His-Cys-Tyr-Glu-Gln-Leu-Val-gly-ser-gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7.1 (Type 6b) [SEQ ID No. 17]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Gly-Leu-His-Cys-Tyr-Glu-Gln-Leu-Val E7.2 (Type 6b)-BiP [SEQ ID No. 18]Pro-Leu-Lys-Gln-His-Phe-Gln-Ile-Val-gly-ser-gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7.2 (Type 6b) [SEQ ID No. 19]His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Pro-Leu-Lys-Gln-His-Phe-Gln-Ile-Val

Hybrid polypeptides for use in vaccines against human papilloma virus ofother types or proteins from other viruses, bacteria etc can bedeveloped by searching their sequences for MHC class I restrictedpeptide epitopes containing the HLA-A2 peptide binding motif.

EXAMPLE 11 Preparation of Recombinant GP96

The DNA sequence encoding a wild-type or KDEL-deleted gp96 polypeptidewas subcloned from pRc/CMV into the vector pET11a (Novagen). Thus uponexpression, mature gp96 could be purified from cell lysates.

Vector Construction

PCR amplification of the sequence encoding gp96 (from pRc/CMV) wasperformed with the following primers. The 5′ primer for both wild-typeand KDEL-deleted gp96 was complementary to the DNA sequence encoding theamino terminal end of the mature form of gp96 and an Nde I restrictionsite (CATATG) the ATG of which forms the initiator codon:

[SEQ ID No. 20] 5′ AGA TAT ACA TAT GGA TGA TGA AGT CGA CGT CC 3′The 3′ primers were complementary to the DNA sequence of gp96 encodingthe carboxyl terminal end of the protein, with the nucleotides encodingthe KDEL sequence removed in the primer for the KDEL-deleted variant.Both primers contain a BamH I restriction site (GGATCC) followed by aSTOP codon as shown:

Wild-type: [SEQ ID No. 21] 5′ TCG CAT CCT TAC AAT TCA TCC TTC TCT GTAGAT TC 3′ KDEL-deleted: [SEQ ID No. 22] 5′ TCG GAT CCT TAC TCT GTA CATTCC TTT TC 3′

The PCR products were cut with Nde I and BamH I and ligated into pET11a(Novagen) which had also been cut with these enzymes. The ligationproduct was used to transform competent BL21 cells. Clones obtained werescreened by expression screening.

Expression and Purification

This procedure is identical for wild-type or KDEL-deleted gp96. Twoliters of E. coli BL21 cells transformed with pET11a containing asequence coding for either wild-type or KDEL-deleted gp96 were grown in2×TY medium supplemented with 200 ug/ml ampicillin at 37° C. until theyreached an absorbance at 600 nm of 0.5-0.6 at which point they wereinduced by the addition of 1 mM IPTG. The cells were allowed to grow fora further 2-5 hours at 37° C. and then they were harvested by 10 minutescentrifugation at 7000×G. The cell pellet was resuspended in 50 mM HepespH 7.5, 50 mM KCl, 5 mM MgAcetate, 20% sucrose, 1 mM PMSF and the cellslysed by passing them through the French Press three times. The cellextract was clarified by a one hour spin at 200000×G and the supernatantretained.

The supernatant was diluted two-fold with cold 50 mM Hepes pH 7.5 andloaded onto a Pharmacia XK26 column containing 50 ml of DE52 anionexchange resin (Whatman) which had been equilibrated in 50 mM Mops pH7.4., 10 mM NaCl, 5 mM MgAcetate. The bound protein was eluted in a0-1000 mM NaCl gradient. Fractions containing gp96 were identified bySDS-PAGE and pooled.

The pooled gp96-containing fractions were diluted two-fold with cold 50mM Mops pH 7.4 and loaded onto a Pharmacia XK16 column containing 15 mLof hydroxylapatite resin (BioRad) which had been washed with 0.5 MK₂HPO₄ pH 7.2., 50 mM KCl and equilibrated in 10 mM K₂HPO₄, pH 7.2, 50mM KCl. The bound protein was eluted in a 10-500 mM K₂HPO₄ pH 7.2gradient with the KCl concentration held constant at 50 mM. Fractionscontaining gp96 were identified by SDS-PAGE and pooled.

The pooled gp96-containing fractions were finally loaded onto aPharmacia XK26 column containing 25 ml of phenyl Sepharose (Pharmacia)which had been equilibrated in 50 mM Mops pH 7.2, 500 mM NaCl and elutedin a 500-0 mM NaCl gradient. The fractions containing essentially puregp96 were pooled, concentrated by filtration and made up to 10%glycerol. The purified gp96 was stored frozen at −80° C.

EXAMPLE 12 Construction of BiP Expression Vector and Purification ofRecombinant BiP

The DNA sequence encoding the wild-type or KDEL-deleted BiP polypeptidewas subcloned from pCDNA3 into the vector pET22 (Novagen), therebyplacing it behind and in frame with a DNA sequence that codes for asignal sequence which targets the expressed BiP to the periplasmic spaceof the bacterial expression host, E. coli. Upon transport into theperiplasm the signal sequence is removed and thus mature wild-type orKDEL-deleted BiP can be harvested from the periplasm without anycontamination by cytosolic hsp70s.

Vector Construction:

PCR amplification of the sequence encoding BiP (from pCDNA3) wasperformed with the following primers. The 5′ primer for both wild-typeand KDEL-deleted BiP was complementary to the DNA sequence of BiPencoding the amino terminal end of the mature form of BiP with an Msc Irestriction site (TGGCCA) immediately upstream from the initiator ATGcodon:

[SEQ ID No. 23] 5′ AGA TAT GTG GCC ATG GAG GAG GAG GAC AAG 3′The 3 primers were complementary to the DNA sequence of BiP encoding thecarboxyl terminal end of the protein, with the nucleotides encoding theKDEL sequence removed in the primer for the KDEL-deleted variant. Bothprimers contain a BamH I restriction site (GGATCC) followed by stopcodon as shown:

Wild type: [SEQ ID No. 24] 5′ TCG GAT CCC TAC AAC TCA TCT TTT TCT G 3′KDEL-deleted: [SEQ ID No. 25] 5′ TCG GAT CCC TAT TCT GAT GTA TCC TCT TCACC 3′

The PCR products were cut with Msc I and BamH I and ligated into pET22(Novagen) which had also been cut with these enzymes. The ligationproduct was used to transform competent BL21 cells. Clones obtained werescreened by expression screening.

Expression and Purification

The procedure is identical for wild-type or KDEL-deleted BiP. Two litersof BL21 cells transformed with pET22 containing a sequence coding foreither wild-type or KDEL deleted BiP were grown in 2×TY mediumsupplemented with 200 μg/ml ampicillin at 37° C. until they reached anabsorbance at 600 nm of 0.5-0.6 at which point they were induced by theaddition of 1 mM IPTG. The cells were allowed to grow for a further 2-5hours at 37° C. and then they were harvested by 10 minutescentrifugation at 7000×G. The cell pellet was gently resuspended in 400mL (or 80 mL/gm cells) of 30 mM Tris pH 8.0, 20% Sucrose, 1 mM PMSF.Following resuspension of the cells EDTA was added to 1 mM and thesuspension incubated at room temperature for 5 minutes. The cells werethen spun down for 15 minutes at 7000×G and resuspended in 400 mL of icecold 5 mM MgSO₄, 1 mM PMSF and incubated at 4° C. for 10 minutes. Thecells were then spun down once again and the supernatant kept since thisnow constitutes the periplasmic extract.

The periplasmic extract was loaded onto a Pharmacia XK26 columncontaining 25 mL of DE52 anion exchange resin (Whatman) which had beenequilibrated in 50 mM Mops pH 7.4, 10 mM NaCl. The bound protein waseluted in a 10-500 mM NaCl gradient. Fractions containing eluted BiPwere identified by SDS-PAGE and pooled. The pooled BiP was subsequentlyrun onto a Pharmacia XK26 column containing 10 mL of ATP agarose whichhad been equilibrated in 50 mM Mops pH 7.4., 100 mM NaCl, 5 mMMgAcetate, 10 mM KCl. After loading the pooled BiP containing fractionsthe column was washed until the baseline of absorption at 280 nm reachedzero. Finally the bound BiP was eluted with the same buffer supplementedwith 1 mM ATP. The eluate was concentrated by filtration, made up to 10%glycerol and stored frozen at −80° C.

EXAMPLE 13 Preparation of Recombinant Mouse HSP40 Plasmid Constructions

The DNA fragment used to introduce an Nde I site at the initiationmethionine of hsp40 was constructed via polymerase chain reaction (PCR)using an

Nde-primer 5′-CCGCAGGAGGGGCATATGGGTAAAGAC-3′ [SEQ ID No. 26] and anNco-primer 5′-GAGGGTCTCCATGGAATGTGTAGCTG-3′. [SEQ ID No. 27]The latter included an Nco I site corresponding to nucleotide 322 of thehuman hsp40 cDNA clone, pBSII-hsp40, Ohtsuka, K., Biochem. Biophys. Res.Commun. 197: 235-240 (1991), which was used as the template. TheHsp40-coding region of pBSII-hsp40 was digested with BamH I and Sac Iand inserted into the complementary sites in a modified form of theplasmid pET-3a (Novagen, Inc.). The PCR-amplified DNA was digested withNde I and Nco I, and replaced the Nde I-Nco I region of the aboveplasmid to create the plasmid pET/hsp40, expressing hsp40.

Protein Purification:

To purify recombinant human hsp40, the plasmid pET/hsp40 was transformedinto BL21(DE3) cells grown at 37° C. After a 2 hour incubation with 0.4mM isopropyl thio-b-D-galactoside (IPTG), cells were lysed in a FrenchPressure Cell (SLM Instruments, Inc.) in buffer A [20 mM Tris-HCl, pH7.5, 20 mM NaCl, 1 mM EDTA] containing 1 mM PMSF. The cleared lysate wasmixed with DEAE-Sephacel (Pharmacia) on ice for 1 h. The unboundmaterial was collected and the resin was washed with buffer A. Theflow-through and first wash were combined and loaded onto ahydroxyapatite HTP column (Bio-Rad) equilibrated with 100 mM potassiumphosphate, pH 7.6. The column was washed with the same buffer and Hsp40was eluted with a linear gradient of 100-300 mM potassium phosphate, pH7.6. Peak fractions were rechromatographed on an HTP column afterpassing them through a DEAE-Sephacel column.

EXAMPLE 14

Vaccine compositions were prepared by combining recombinant mouse hsp70(prepared as in example 2), recombinant human hsp40 (prepared as inexample 13) and Ova-peptide

Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu [SEQ ID NO. 7]in a final volume of 10 μl of buffer (20 mM Hepes pH 7.0, 150 mM KCl, 10mM (NH₄)₂SO₄, 2 mM MgCl₂ and 2 mM MgATP) as follows:

Sample hsp70 hsp40 ova OVA-alone nil nil 5 ug Hsp70/40 15 ug 8 ug nilHsp70/40 + 15 ug 8 ug 5 ug OVA Hsp70 + OVA 15 ug 5 ugThe mixtures were incubated for 30 minutes at 37° C. prior to use forimmunizations.

C57BL/6 mice were immunized intradermally once a week for a total of twoweeks with 10 uL of one of the mixtures described above or with amixture of TiterMax® (Vaxcell, Norcross, Ga.) and Ova-peptide (5 ug).One week after the second immunization, spleen cells were removed andmononuclear cells (6-8×10⁷) were cultured with 3×10⁶ γ-irradiated (3000rad) stimulator cells. The stimulator cells were obtained from naivemice that had been sensitized in vitro with ova peptide (10 mg/ml) for30 minutes at room temperature, washed and irradiated at 3000 rads.

Cytotoxicity of spleen cells from vaccinated mice was assayed onOva-peptide pulsed EL4 cells in an 18-hour chromium release assay. CTLwere generated by culturing in vivo immunized spleen cells for 5 days ata concentration of 10⁶ cells/mL in RPMI medium, 10% FCS, penicillin,streptomycin and 2 mM L-glutamine, together with 3×10⁶ γ-irradiated(3,000 rad) stimulator cells/mL. Target cells were prepared by culturingcells for 1 hour in the presence of 250 uCi of ⁵¹Cr sodium chromate(DuPont, Boston, Mass.) in Tris-phosphate buffer, pH 7.4 at 37° C. for60 minutes. After washing, 10⁴ ⁵¹Cr-labeled target cells were mixed witheffector lymphocytes to yield several different effector/target (E/T)ratio and were incubated for 18 hours. Supernatants were harvested andthe radioactivity was measured in a gamma counter. Percent specificlysis was calculated as: 100×[Cpm release by CTL−cpm spontaneousrelease)/(cpm maximal release−cpm spontaneous release)]. Maximalresponse was determined by addition of 1% Triton X-100. Spontaneousrelease of all target in the absence of effector cells was less than 25%of the maximal release.

The results of this study are shown in FIG. 8. As shown, combinations ofantigen with hsp70 or a mixture of hsp70 and hsp40 are effective toproduce a CTL response to the antigen, while the administration of theantigen alone or a combination of heat shock proteins is not.

EXAMPLE 15

The experiment of Example 14 was repeated using EG7 lymphoma cells,Moore et al., Cell 54: 777-785 (1988), in place of the EL4 cells. Theresults are shown in FIG. 9 and are comparable to those observed usingEL4 cells.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1-40. (canceled)
 41. A composition comprising (a) isolated complexeseach comprising a hybrid antigen non-covalently bound to a heat shockprotein, wherein the hybrid antigen comprises an immunogenic domain anda heat shock protein binding domain, wherein the immunogenic domain iscovalently linked to the heat shock protein binding domain; wherein theheat shock protein binding domain is a peptide, and wherein theimmunogenic domain comprises an epitope of an antigen of: a neoplasticdisease, an infectious agent of an infectious disease, an autoimmunedisease or an allergy; and (b) a pharmaceutically acceptable carrier.42. The composition of claim 41, wherein said immunogenic domaincomprises a protein or peptide antigen, or a selected epitope of saidprotein or peptide antigen.
 43. The composition of claim 42, whereinsaid immunogenic domain is 7-20 amino acids.
 44. The composition ofclaim 42, wherein said immunogenic domain is 4-100 amino acids.
 45. Thecomposition of claim 41 or 42, wherein said immunogenic domain comprisesan epitope associated with the activation of B cells, T helper cells orcytotoxic T cells.
 46. The composition of claim 41, wherein saidimmunogenic domain comprises a carbohydrate, a nucleic acid or a lipid.47. The composition of claim 41, wherein the heat shock protein isprepared using recombinant techniques.
 48. The composition of claim 41,42 or 44, wherein said heat shock protein binding domain is 7-15 aminoacids.
 49. The composition of claim 45, wherein said heat shock proteinbinding domain is 7-15 amino acids.
 50. The composition of claim 41,wherein the heat shock protein is selected from the group consisting ofBiP, hsp70, hsc70, gp96, hsp60, hsp40 and hsp90.
 51. The composition ofclaim 41, wherein the heat shock protein is hsp70.
 52. The compositionof claim 41, wherein the heat shock protein is hsc
 70. 53. Thecomposition of claim 41, wherein the heat shock protein is gp96.
 54. Thecomposition of claim 41, wherein the heat shock protein is BiP.
 55. Thecomposition of claim 41, wherein said heat shock protein binding domaincomprises the amino acid sequence His Trp Asp Phe Ala Trp Pro Trp (SEQID NO:1).
 56. The composition of claim 41, wherein said heat shockprotein binding domain comprises the amino acid sequence Phe Trp Gly LeuTrp Pro Trp Glu (SEQ ID NO:4), Gln Lys Arg Ala Ala (SEQ ID NO:5) or ArgArg Arg Ala Ala (SEQ ID NO:6).
 57. The composition of claim 41, whereinsaid immunogenic domain and said heat shock protein binding domain areseparated by a peptide linker.
 58. The composition of claim 57, whereinthe peptide linker is Gly Ser Gly.
 59. The composition of claim 41,wherein the hybrid antigen is a peptide.
 60. The composition of claim41, wherein the isolated complexes comprise different heat shockproteins.
 61. The composition of claim 41, 44, 50, or 51, wherein theisolated complexes comprise different hybrid antigens.
 62. Thecomposition of claim 41, wherein the immunogenic domain comprises anepitope of an antigen of a neoplastic disease, or an epitope of anantigen of an infectious agent of an infectious disease.
 63. Thecomposition of claim 61, wherein the immunogenic domain comprises anepitope of an antigen of a neoplastic disease, or an epitope of anantigen of an infectious agent of an infectious disease.
 64. Thecomposition of claim 41, wherein the immunogenic domain comprises anepitope of an antigen of an autoimmune disease or allergy.
 65. Thecomposition of claim 62, wherein the immunogenic domain comprises anepitope of an antigen of a neoplastic disease.
 66. The composition ofclaim 65, wherein the neoplastic disease is selected from the groupconsisting of a sarcoma, lymphoma, leukemia and carcinoma.
 67. Thecomposition of claim 66, wherein the neoplastic disease is selected fromthe group consisting of melanoma, carcinoma of the breast, carcinoma ofthe prostate, ovarian carcinoma, carcinoma of the cervix, uterinecarcinoma, colon carcinoma, carcinoma of the lung, glioblastoma, andastrocytoma.
 68. The composition of claim 62, wherein the immunogenicdomain comprises an epitope of an antigen of an infectious agent of aninfectious disease.
 69. The composition of claim 68, wherein theinfectious agent is a bacterium, virus, protozoan, mycoplasma, fungus,yeast, parasite or prion.
 70. The composition of claim 68, wherein theinfectious agent is a bacterium.
 71. The composition of claim 68,wherein the infectious agent is a virus.
 72. The composition of claim68, wherein the infectious agent is a protozoan.
 73. The composition ofclaim 70, wherein the bacterium is selected from the group consisting ofSalmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium,Escherichia, Klebsiella, Vibrio, Mycobacterium, and Mycoplasmapneumoniae.
 74. The composition of claim 71, wherein the virus isselected from the group consisting of a human papilloma virus, herpesvirus, retrovirus, hepatitis virus, influenza virus, rhinovirus,respiratory syncytial virus, cytomegalovirus, adenovirus, herpes simplexvirus, herpes zoster virus, human immunodeficiency virus 1, and humanimmunodeficiency virus
 2. 75. The composition of claim 72, wherein theprotozoan is selected from the group consisting of an amoeba, a malarialparasite, and Trypanosoma cruzi.
 76. The composition of claim 41, 44, 50or 62, wherein the heat shock protein is a human heat shock protein. 77.The composition of claim 61, wherein the heat shock protein is a humanheat shock protein.
 78. The composition of claim 41, 44, 50 or 62further comprising one or more adjuvants.
 79. The composition of claim61 further comprising one or more adjuvants.
 80. A method for inducingan immune response to an immunogenic domain in a subject in needthereof, comprising administering, to the subject, the composition ofany one of claims 41, 44, 50, or 62, whereby an immune response to saidimmunogenic domain is induced.
 81. A method for inducing an immuneresponse to an immunogenic domain in a subject in need thereof,comprising administering, to the subject, the composition of claim 61,whereby an immune response to said immunogenic domain is induced.
 82. Amethod for treating an infectious disease in a subject in need thereof,comprising administering, to the subject, a therapeutically effectiveamount of the composition of claim
 68. 83. A method for preventing aninfectious disease in a subject in need thereof, comprisingadministering, to the subject, a prophylactically effective amount ofthe composition of claim
 68. 84. A method for treating a neoplasticdisease in a subject in need thereof, comprising administering, to thesubject, a therapeutically effective amount of the composition of claim65.
 85. A method for preventing a neoplastic disease in a subject inneed thereof, comprising administering, to the subject, aprophylactically effective amount of the composition of claim
 65. 86. Amethod for treating a neoplastic disease in a subject in need thereof,comprising administering, to the subject, a therapeutically effectiveamount of the composition of claim 63, and wherein said immunogenicdomain comprises an antigen of said neoplastic disease.
 87. A method forpreventing a neoplastic disease in a subject in need thereof, comprisingadministering, to the subject, a prophylactically effective amount ofthe composition of claim 63, and wherein said immunogenic domaincomprises an epitope of an antigen of said neoplastic disease.
 88. Themethod of claim 80, wherein said administering is repeated at leastonce.
 89. The method of claim 82, 83, 84 or 85 wherein saidadministering is repeated at least once.
 90. The method of claim 80,wherein said subject is a human.
 91. The method of claim 82, 83, 84 or85 wherein said subject is a human.